The present invention relates to new glycerolipid compounds and new compositions containing them. More particularly, the present invention relates to the use of said compounds or of said compositions to prepare a vector for transferring an active substance, in particular a therapeutically active substance comprising negative charges, in particular a polynucleotide, into a target cell, particularly a vertebrate cell, and more particularly a mammalian cell.
The transfer of a gene into a given cell is the very basis of gene therapy. This new technology, whose field of application is vast, makes it possible to envisage the treatment of serious diseases for which the conventional therapeutic alternatives are not very effective, or are even inexistent, and applies to diseases which are either of genetic origin (hemophilia, cystic fibrosis, myopathy and the like) or acquired (cancer, AIDS and the like).
During the past 30 years, numerous tools have been developed which allow the introduction of various heterologous genes into cells, in particular mammalian cells. These different techniques may be divided into two categories. The first category relates to physical techniques such as microinjection, electroporation or particle bombardment which, although effective, are greatly limited to applications in vitro and whose implementation is cumbersome and delicate. The second category involves techniques relating to molecular and cell biology in which the gene to be transferred is combined with a vector of a biological or synthetic nature which promotes the introduction of said material.
Currently, the most effective vectors are viral, in particular adenoviral or retroviral, vectors. The techniques developed are based on the natural properties which these viruses have to cross the cell membranes, to escape degradation of their genetic material and to cause their genome to penetrate into the nucleus. These viruses have already been the subject of numerous studies and some of them are already used experimentally as vectors for genes in humans for the purpose, for example, of a vaccination, an immunotherapy or a therapy intended to make up for a genetic deficiency. However, this viral approach has many limitations, in particular because of the limited capacity for cloning into the viral genome, the risks of spreading in the host organism and in the environment the infectious viral particles produced, the risk of artefactual mutagenesis by insertion into the host cell in the case of retroviral vectors, and the high induction of immune and inflammatory responses in vivo during the therapeutic treatment, considerably limiting the number of administrations which can be envisaged (McCoy et al., 1995, Human Gene Therapy, 6, 1553-1560; Yang et al., 1996, Immunity, 1, 433-442). These numerous disadvantages, in particular in the context of a use in humans, have led several teams to develop alternative systems of transferring polynucleotides.
Several non-viral methods are currently available. By way of example, there may be mentioned coprecipitation with calcium phosphate, the use of receptors mimicking viral systems (for a review see Cotten and Wagner, 1993, Current Opinion in Biotechnology, 4, 705-710), or the use of polymers such as polyamidoamine (Haensler and Szoka, 1993, Bioconjugate Chem., 4, 372-379) or of polymer such as those presented in WO 95/24221 describing the use of dendritic polymers, the document WO 96/02655 describing the use of polyethyleneimine, or of polypropyleneimine and the documents U.S. Pat. No. 5,595,897 and FR 2,719,316 describing the use of conjugates of polylysine. Other non-viral techniques are based on the use of liposomes whose value as agent allowing the introduction, into cells, of certain biological macromolecules, such as for example DNA, RNA, proteins or certain pharmaceutically active substances, has been widely described in the literature. To this end, several teams have already proposed the use of cationic lipids which have a high affinity for the cell membranes and/or the nucleic acids. Indeed, although it has been shown, in the case of nucleic acids, that this type of macromolecule is capable of crossing the plasma membrane of some cells in vivo (WO 90/11092), it is nevertheless the case that the observed transfection efficiency is still highly limited, because of in particular the polyanionic nature of the nucleic acids which prevent their passage across the cell membrane, which itself has a negative net apparent charge. Since 1989 (Felgner et al., Nature, 337, 387-388), cationic lipids have been presented as molecules which are advantageous for promoting the introduction of large anionic molecules, such as nucleic acids, into certain cells. These cationic lipids are capable of complexing anionic molecules, thus tending to neutralize the negative charges on said molecules and to promote their coming close to the cells. Many teams have already developed various cationic lipids. By way of example, there may be mentioned DOTMA (Felgner et al., 1987, PNAS, 84, 7413-7417), DOGS or Transfectam(trademark) (Behr et al., 1989, PNAS, 86, 6982-6986), DMRIE and DORIE (Felgner et al., 1993, Methods 5, 67-75), DC-CHOL (Gao et Huang, 1991, BBRC, 179, 280-285), DOTAP(trademark) (McLachlan et al., 1995, Gene Therapy, 2,674-622) or Lipofectamine(trademark), as well as those described in Patent Applications WO9116024 or WO9514651.
More particularly, Application WO9405624 describes cationic lipids of formula: 
in which the R radicals are in particular octadecenyl radicals, the Z radicals are C1-C13 alkyl or xe2x80x94C(xe2x95x90O)xe2x80x94(C1-C13) alkyl or acyl radicals, q is an integer from 1 to 6, X is in particular a short polyamine chain, such as spermine, spermidine, carboxyspermine or polylysine.
Applications EP 685457 and EP 685234 describe in particular cationic compounds of formula: 
in which R is in particular a hydrocarbon chain having 10 to 30 carbon atoms, saturated or otherwise, A may in particular be chosen from the groups xe2x80x94Oxe2x80x94(Cxe2x95x90O)xe2x80x94 and xe2x80x94NHxe2x80x94(Cxe2x95x90O)xe2x80x94, n varies from 0 to 4 and R1 is an alkyl or a short aminoalkyl chain in which the primary amine is substituted with an alkyl having 2 to 8 carbon atoms. These compounds have a low hemolytic activity and make it possible in vitro to introduce into HelaS3 cells a double-stranded RNA capable of acting as growth inhibiting agent.
Application DE 19521412 describes compounds which also comprise a nonpolar part and a polar part of formula: 
in which p varies from 1 to 6, q varies from 0 to 2, R is either C(xe2x95x90O)xe2x80x94C1-23 or C1-23, saturated or otherwise, and Z is a peptide, an aminoacid or a branched amino structure. These cationic compounds allow the in vitro transfection of cells in culture.
However, several studies (by way of example, see Mahato et al., J. Pharm. Sci., 1995, 84, 1267-1271, Thierry et al., 1995, P.N.A.S., 92, 9742-9746) have demonstrated that the efficiency of transferring the anionic macromolecule into cells could vary depending in particular on the interaction between the complexes and the cell membranes, the cell considered, the lipid composition of the cationic compounds, the size of the complexes formed with the anionic molecules and more particularly the ratio between the positive and negative charges on the different components of said complex. The mechanisms which allow in particular the interaction of the complexes with the cell membranes and the transfer of the complexes into the cell are still to a large extent poorly understood and researchers proceed in their studies based on a highly empirical approach. It is consequently desirable to provide other cationic lipids possibly having improved properties or properties which are different from the cationic lipids already described.
The Applicant has now identified new glycerolipid compounds, which can be provided in cationic form, useful in particular for transferring an active substance comprising negative charges, in particular a polynucleotide, into a target cell, whose use may be envisaged in particular in vivo in the context of a gene therapy.
Accordingly, the subject of the present invention is first of all a compound of formula: 
in which:
R1 and R2, which are identical or different, are alkyl or alkenyl radicals having 6 to 23 carbon atoms (noted C6-C23), which are linear or branched, or radicals xe2x80x94C(xe2x95x90O)xe2x80x94(C6-C23) alkyl or xe2x80x94C(xe2x95x90O)xe2x80x94(C6-C23) alkenyl, or more particularly xe2x80x94C(xe2x95x90O)xe2x80x94(C12-C20) alkyl or xe2x80x94C(xe2x95x90O)xe2x80x94(C12-C20) alkenyl, which are linear or branched, aryl radicals, cycloalkyl radicals, fluoroalkyl radicals, oxyethylene or oxymethylene groups which are optionally repeated, linear or branched, optionally substituted,
X is an oxygen atom or an amino radical xe2x80x94NR3, R3 being a hydrogen atom or an alkyl radical having 1 to 4 carbon atoms,
n is a positive integer from 1 to 6, preferably from 2 to 4,
m is a positive integer from 1 to 6, preferably from 2 to 4, and when n greater than 1, m may be identical or different from said n.
The term xe2x80x9calkenylxe2x80x9d is intended to indicate that the carbon chain in question may comprise one or more double bond(s) along said chain.
According to a specific case of the invention, said compounds are characterized in that R1 and/or R2 are fluorinated, that is to say that at least one carbon of the polycarbon chain is substituted by a fluorinated group. Examples of such molecules are provided in Example N. In this specific case, the number of fluorinated carbon atoms on each chain R1 or R2 may vary from 1 to 12, and more particularly from 4 to 8, and is preferably 4. According to an advantageous case, R1 and/or R2 are alkyl radicals having 15 carbon atoms and the number of fluorinated carbon atoms is 4 for each of the relevant chains R1 and R2. The number of fluorinated groups present on the chains R1 and/or R2 may in particular vary from 1 to 23, more particularly from 9 to 17 and preferably is 9.
The compounds according to the invention may, in addition, be substituted. Such substitutions may in particular consist of a labeling molecule (see labeling molecules in U.S. Pat. No. 4,711,955) which makes it possible, for example, to visualize the distribution of the compounds or of the complexes containing them after administration in vitro or in vivo, a cell targeting molecule or an anchoring molecule. The invention consequently also relates to a compound as presented above, conjugated with one or more targeting components via the intermediacy of at least a) one of the carbon atoms, in particular chosen from those present on the groups R1, R2 and/or R3 or b) one of the secondary or primary nitrogen atoms of the polyamine chain. Such components may allow targeting to a specific cell type, facilitate penetration into the cell, lysis of the endosomes or alternatively intracellular transport and are widely described in the literature. They may be, for example, all or part of sugars, peptides (GRP peptide, Gastrin Releasing Peptide, for example), oligonucleotides, lipids, hormones, vitamins, antigens, antibodies, ligands specific for membrane receptors, ligands capable of reacting with an anti-ligand, fusogenic peptides, nuclear localization peptides, or a combination of such compounds. There may be mentioned more particularly the galactosyl residues which make it possible to target the asyaloglycoprotein receptor at the surface of hepatic cells, the fusogenic peptide INF-7 derived from the influenza virus hemagglutinin subunit HA-2 (Plank et al., 1994, J. Biol. Chem. 269, 12918-12924) or a nuclear localization signal derived from the SV40 virus T antigen (Lanford and Butel, 1984, Cell 37, 801-813) or the Epstein Barr virus EBNA-1 protein (Ambinder et al., 1991, J. Virol. 65, 1466-1478).
Such conjugates can be easily obtained by techniques widely described in the literature, and more particularly by chemical coupling, in particular using protecting groups such as trifluoroacetyl or Fmoc or Boc, onto the polyamine. The selective deprotection of a protecting group then makes it possible to couple the targeting component, and the glycerolipid is then deprotected. It should be stated, however, that the substitution of the nonreactive groups such as the carbon atoms in the CH or CH2 groups will be carried out during synthesis of the compounds of the invention by methods known to a person skilled in the art, whereas the reactive groups, such as the primary or secondary amines, may be the subject of substitutions on the neosynthesized glycerolipids of the invention.
According to an advantageous case of the invention, said compound is in a cationic form, that is to say that it is in a form which is protonated by binding of a proton onto one or more nitrogen atoms present on the polyamine chain. In this case, said cationic glycerolipid is combined with one or more biologically acceptable anions, such as for example the trifluoroacetate, halide, monomethylsulfate, acetate or phosphate, iodide, chloride, or bromide anion and the like. It is also possible to obtain compounds in cationic form by substitution of the amines, for example, with a methyl or ethyl radical, and the like.
The compounds according to the present invention comprise from 2 to 7 positive charges, more particularly from 3 to 5, and preferably 5. It has been shown that the affinity of a polyamine for DNA depends in particular on the number of amine functional groups present on said polyamine (Braulin, W. H., Strick, T. J., and Record, M. T., Jr. (1982) Biopolymers 21, 1301-1314). Moreover, following their penetration into the cell by endocytosis, the complexes formed between a DNA and a cationic lipid compound are located in the endosomes in which the DNA may be degraded under the action of pH-dependent nucleases. To counter this phenomenon which affects the transfection efficiency, it is possible to use lysosomotropic agents, such as for example chloroquine, whose buffering capacity at pH 5.5 makes it possible to observe an improvement in the transfection. However, the efficiency of such compounds is not systematic; there may be noted, by way of negative examples, the cases of polyethyleimine (PEI), lipospermines or dendrimers of polyamidoamine (PAMAM). Moreover, the use of chloroquine at a high dose may present a toxic risk. According to the invention, the compounds possess a polyamine head which makes it possible to obtain a similar effect to that of the lysosomotropic molecules. A specific advantage of said compounds might consequently be to avoid the use of chloroquine for the in vivo applications.
According to a preferred embodiment of the invention, said compound is chosen from the group consisting of the following formulae: 
These cationic compounds may, for example, be prepared by reacting a compound of formula: 
in which:
R4 and R5 are protecting groups forming in particular together an isopropylidene radical,
X has the same meaning as in formula I, with an acid of formula: 
m and n having the same meaning as in formula I,
R6 being a protecting group, in particular t-butoxycarbonyl (BOC).
The functional groups Oxe2x80x94R4 and xe2x80x94Oxe2x80x94R5 are then deprotected so as to attach by esterification or etherification the radicals R1 and R2 in a known manner.
The compound obtained is deprotected in the presence of trifluoroacetic acid. The acid of formula VII is prepared in a known manner.
In the case of the compounds of the invention for which m=3, n=2 or 3, reference will be made to the examples indicated below in order to know the practical modalities for the synthesis. The processes described are applicable in general to the syntheses of the compounds according to the invention subject to adaptations within the capability of persons skilled in the art. However, the compounds of the invention cannot be limited to those obtained by the modes of preparation described above.
According to another aspect, the invention also relates to a composition comprising at least one compound as described above and optionally at least one adjuvant capable of enhancing the formation of the complex between a said compound and an active substance, or of enhancing the function of these complexes toward the cell.
Preferably, such an adjuvant will be a neutral or zwitterionic lipid which, for example, is or is derived from a triglyceride, a diglyceride, cholesterol (see for example U.S. Pat. No. 5,438,044), in particular, a neutral or zwitterionic lipid which is or is derived from a phosphatidylethanolamine (PE), phosphatidylcholine, phosphocholine, sphyngomyelin, ceramide or cerebroside. Advantageously, dioleoylphosphatidylethanolamine (DOPE) will be chosen.
The weight ratio between the compound of the invention and the neutral or zwitterionic lipid is generally between 0.1 and 10, it being understood that this ratio may vary depending on the nature of the components considered. Persons skilled in the art have sufficient knowledge to allow these minor adaptations. It is also possible to use a mixture of neutral and/or zwitterionic lipids.
The invention relates, in addition, to a complex comprising at least one compound or at least one composition as described above and at least one active substance, in particular a therapeutically active substance, comprising at least one negative charge. According to a variant of the invention, said complex may, in addition, contain one or more cationic amphiphilic agents such as those described in the literature of which examples were provided above. In general, there will be used therapeutically active substances which may be used in particular in the context of gene therapy.
According to a specific embodiment, said active substance is chosen from nucleic acids and proteins. Preferably, the active substance of the complex according to the invention is a polynucleotide, said compound or said composition then making it possible to enhance the transfecting power of the polynucleotide in a cell.
xe2x80x9cPolynucleotidexe2x80x9d is understood to designate a DNA and/or RNA fragment which is double-stranded or single-stranded, linear or circular, natural, isolated or synthetic, designating a precise succession of nucleotides, which are modified or otherwise (see by way of example U.S. Pat. No. 5,525,711), labeled or otherwise (see for example U.S. Pat. No. 4,711,955 or EP 302175), making it possible to define a fragment or a region of a nucleic acid without size limitation. Polynucleotide is understood to designate in particular a cDNA, a genomic DNA, a plasmid DNA, a messenger RNA, an antisense RNA, a ribozyme, a transfer RNA, a ribosomal RNA or a DNA encoding such RNAs. xe2x80x9cPolynucleotidexe2x80x9d or xe2x80x9cnucleic acidxe2x80x9d are synonymous terms in the context of the present application.
According to a specific embodiment of the invention, said polynucleotide comprises a gene of interest and components allowing the expression of said gene of interest. In this embodiment, said polynucleotide is advantageously in the form of a plasmid. The components allowing expression are all the components allowing the transcription of said DNA fragment into RNA (antisense RNA or mRNA) and the translation of the mRNA into a polypeptide. They are in particular promoter sequences and/or regulatory sequences which are effective in said cell, and optionally the sequences required to allow excretion or expression of said polypeptide at the surface of the target cells. By way of example, there may be mentioned promoters such as the promoters of the viruses RSV, MPSV, SV40, CMV or 7.5k, of the vaccinia virus, the promoters of the gene encoding muscle creatine kinase, actin, or pulmonary surfactant. It is, in addition, possible to choose a promoter sequence specific for a given cell type or which can be activated under defined conditions. The literature provides a large amount of information relating to such promoter sequences. Moreover, said polynucleotide may comprise at least two sequences, which are identical or different, exhibiting a transcriptional promoter activity and/or at least two coding DNA sequences, which are identical or different, situated, relative to each other, contiguously, far apart, in the same direction or in the opposite direction, as long as the transcriptional promoter function or the transcription of said sequences is not affected. Likewise, it is possible to introduce into this type of nucleic acid construct xe2x80x9cneutralxe2x80x9d nucleic sequences or introns which do not affect transcription and are spliced before the translation step. Such sequences and their uses are described in the literature. Said polynucleotide may also contain sequences required for intracellular transport, for replication and/or for integration. Such sequences are well known to persons skilled in the art. Moreover, the polynucleotides according to the present invention may also be polynucleotides which are modified such that it is not possible for them to become integrated into the genome of the target cell or polynucleotides which are stabilized with the aid of agents such as, for example, spermine.
In the context of the present invention, the polynucleotide may be homologous or heterologous to the target cell. It may be advantageous to use a polynucleotide which encodes all or part of a polypeptide, in particular a polypeptide having a therapeutic or prophylactic activity, and more particularly an immunogenic activity of the cellular or humoral type. The term polypeptide is understood without restriction as to its size or its degree of modification (for example glycosylation). There may be mentioned, by way of example, the genes encoding an enzyme, a hormone, a cytokine, a membrane receptor, a structural polypeptide, a polypeptide forming a membrane channel, a transport polypeptide, an adhesion molecule, a ligand, a factor for regulation of transcription, of translation, of replication, or of the stabilization of the transcripts, or an antibody, such as for example the gene encoding the CFTR protein, dystrophin, factor VIII or IX, E6/E7 of HPV, MUCl, BRAC1, xcex2-interferon, xcex3-interferon, interleukin (IL)2, IL-4, IL-6, IL-7, IL-12, tumor necrosis factor (TNF) type alpha, GM-CSF (Granulocyte Macrophage Colony Stimulating Factor), the Herpes Simplex virus type 1 (HSV-1) tk gene, the gene associated with retino-blastoma or p53 or all or part of immunoglobulins, such as the fragments F(ab)2, Fabxe2x80x2, Fab or the anti-idiotypes (U.S. Pat. No. 4,699,880). This list is of course not limiting and other genes may be used.
According to a preferred embodiment, the complexes according to the invention are small in size (less than 500 nm, advantageously less than 200 nm and preferably less than 100 nm).
Moreover, the transfection experiments carried out show that advantageously the weight ratio of the lipid compound according to the invention to said polynucleotide is 0.01 to 100. The optimum ratio is between 0.05 and 10.
The invention also relates to a process for preparing the complexes cationic compounds/active substances comprising at least one negative charge, said process being characterized in that one or more compounds or compositions according to the invention are brought into contact with one or more active substances comprising at least one negative charge and in that said complex is recovered, optionally after a purification step.
In a first instance, according to a first variant, one or more cationic compounds are dissolved with an appropriate quantity of solvent or mixture of solvents which are miscible in water, in particular ethanol, dimethylsulfoxide (DMSO), or preferably a 1:1 (v:v) ethanol/DMSO mixture, so as to form lipid aggregates according to a known method described, for example, in Patent Application WO-A-9603977, or according to a second variant, are suspended with an appropriate quantity of a solution of detergent such as an octylglucoside such as n-octyl xcex2-glucopyranoside, or 6-O-(N-heptylcarbamoyl)-methyl-xcex2-D-glucopyranoside.
The suspension may then be mixed with a solution of active substance comprising negative charges.
In the case where it is desirable that a neutral or zwitterionic lipid is present in the final complex, a film is formed, in the known manner, prior to the dissolution in the solvent which is miscible with water or in the solution of detergent, with a mixture containing a said cationic compound and a said neutral or zwitterionic lipid, such as for example DOPE.
One of the important characteristics of the process consists in the choice of the ratio between the positive charges of the cationic lipid and the negative charges of the active substance.
Without wishing to be limited by a specific ratio, quantities of the different charges will be chosen so that the ratio between the number of positive charges of the cationic compound or composition and the number of negative charges of the active substance is between 0.05 and 20, in particular between 0.1 and 15, and preferably between 5 and 10.
The calculation to arrive at such a ratio will take into consideration the negative charges carried by the active substance and the quantity of compound necessary to satisfy the ratio indicated above will be adjusted. The quantities and the concentrations for the other components are adjusted according to their respective molar masses and the number of their positive and/or negative charges.
This charge ratio also constitutes an advantageous characteristic of the complex according to the invention.
In the case of the second variant and optionally, subsequent dialysis may be carried out in order to reduce the detergent and to recover the complexes. The principle of such a method is for example described by Hofland et al. (1996, PNAS 93, p 7305-7309) and in chapter II of the Philippot et al. document (G. Gregoriadis, 81-89, CRC Press 1993).
It has been shown that the first variant leads to excellent results in terms of the size of the complexes obtained.
According to a third variant, one or more cationic compositions or compounds are suspended in a buffer and then the suspension is subjected to sonication until visual homogeneity is obtained. The lipid suspension is then extruded through two microporous membranes under appropriate pressure. The lipid suspension is then mixed with a solution of active substance comprising negative charges. This so-called sonication-extrusion technique is well known in the art.
The use of a neutral or zwitterionic lipid, such as DOPE, may prove advantageous for the production of complexes which are small in size (less than 200 nm, preferably less than 100 nm).
The characteristics of the complexes formed may be evaluated by several means which make it possible to determine, for example:
the state of complex formation with the active substance, in particular by identification of the free nucleic acids by agarose gel electrophoresis in the case where the substances are nucleic acids,
the size of the particles by a quasi-elastic scattering of light,
the absence of precipitation over the long term.
The object of the present invention is also the complexes obtained using the processes listed above.
The invention also relates to the use of a compound, of a composition or of a complex according to the invention to transfer at least one active substance, especially a therapeutically active substance, in particular a nucleic acid, into target cells, in vitro, ex vivo or in vivo, more particularly in vivo.
xe2x80x9cTarget cellsxe2x80x9d according to the invention is understood to mean prokaryotic cells, yeast cells and eukaryotic cells, plant cells, human or animal cells, and in particular mammalian cells. Cancer cells should, moreover, be mentioned. In vivo, the invention may be applied at the level of the interstitial or luminal space of tissues such as the lungs, trachea, skin, muscle, brain, liver, heart, spleen, bone marrow, thymus, bladder, lymph, blood, pancreas, stomach, kidney, ovaries, testicles, rectum, peripheral or central nervous system, eyes, lymphoid organs, cartilages and endothelium. According to an advantageous choice of the invention, the target cell will be a muscle cell, a hematopoietic stem cell or alternatively a cell of the airways, more particularly a tracheal or pulmonary cell, and advantageously a cell of a respiratory epithelium.
The invention also relates to a process for transferring in vitro a therapeutically active substance into a target cell according to which said cell is brought into contact with a complex according to the invention.
The complexes according to the invention can be used as a medicament for curative, preventive or vaccinal purposes. Accordingly, the subject of the invention is also the complexes of the invention as a medicament for curative, preventive or vaccinal purposes. Such complexes may be used in a method of therapeutic treatment which consists in transferring at least one therapeutically active substance, in particular a polynucleotide, into target cells, in particular a mammalian cell, and more precisely a muscle cell, a hematopoietic stem cell, a cell of the airways, more particularly a tracheal or pulmonary cell, a cell of the respiratory epithelium.
More widely, the present invention also relates to a process for introducing an active substance comprising negative charges into a cell, characterized in that cells cultured on an appropriate medium are brought into contact with a suspension of complexes cationic compound/active substance comprising negative charges. After a certain incubation time, the cells are washed and recovered. The introduction of the active substance may be checked (optionally after lysis of the cell) by any appropriate means.
The process of introduction is well known per se. The term xe2x80x9cintroductionxe2x80x9d is understood to mean that the active substance comprising negative charges is transferred into the cell and is located, at the end of the process, inside said cell or at the level of the membrane thereof. In the case where the active substance is a nucleic acid, reference will be made more particularly to xe2x80x9ctransfectionxe2x80x9d. In this case, the verification of the transfection of the nucleic acid can be carried out by any appropriate means, for example by measuring the expression of the gene considered or the concentration of the expressed protein.
The invention relates more particularly to the use of a compound, of a composition or of a complex according to the invention for the preparation of a medicament for curative, preventive or vaccinal purposes, intended for the treatment of the human or animal body, in particular by gene therapy.
According to a first possibility, the medicament may be administered directly in vivo (for example into a muscle, into the lungs by aerosol and the like). It is also possible to adopt the ex vivo approach which consists in collecting cells from the patient (bone marrow stem cells, peripheral blood lymphocytes, muscle cells and the like), transfecting them in vitro according to the present invention and readministering them to the patient.
The complexes according to the invention may be administered by the intramuscular, intratracheal, intranasal, intracerebral, intrapleural, intratumoral, intracardiac, intragastric, intraperitoneal, epidermal, intravenous or intraarterial route by a syringe or by any other equivalent means, systems suitable for the treatment of the airways or of the mucous membranes such as inhalation, instillation or aerosolization. There may also be mentioned the modes of administration by the topical route, such as for example by application of a cream, by oral administration or any other means perfectly known to the person skilled in the art and applicable to the present invention.
It is also within the scope of the invention to target specific organs or tissues by administration, in particular by the intravenous route, of a complex according to the invention prepared so as to adjust the ratio compound or composition/therapeutically active substance in said complex, the apparent charge of the complex (see in particular Liu et al., 1997, Gene Therapy, 4, 517-523; Thierry et al., 1995, P.N.A.S., 92, 9742-9746).
The invention also relates to a method of gene therapy consisting in administering to a patient an appropriate quantity of a composition according to the invention. According to the present invention and in the context of gene therapy in vivo, it is possible to repeat several times, in a given patient, the method as proposed without any major immune reaction being elicited against one of the compounds administered. The administration may take place in a single dose or repeated once or several times after a certain time interval. The repeated administration would make it possible to reduce the quantity of therapeutically active substance, more particularly of DNA, to be administered for a given dose. The appropriate route of administration and dosage vary according to various parameters, for example the individual or disease to be treated or alternatively the polynucleotide to be transferred.
The invention relates more particularly to a pharmaceutical preparation comprising at least one complex as described above, optionally containing, in addition, at least one adjuvant capable of stabilizing said pharmaceutical preparation for the purpose of its storage for example and/or of enhancing the transfecting power of said complex. Such an adjuvant could, for example, be chosen from the group consisting of chloroquine, a protic polar compound chosen in particular from propylene glycol, polyethylene glycol, glycerol, ethanol, 1-methyl-L-2-pyrrolidone or derivatives thereof, or an aprotic polar compound chosen in particular from dimethyl sulfoxide (DMSO), diethyl sulfoxide, di-n-propyl sulfoxide, dimethyl sulfone, sulfolane, dimethylformamide, dimethylacetamide, tetramethylurea, acetonitrile or derivatives thereof. Likewise, said preparation may contain a pharmaceutically acceptable carrier allowing its administration to humans or animals.
In the context of the use of a method of treatment in vivo according to the present invention, it is, in addition, possible to carry out, before the administration of a pharmaceutical preparation as described above, a treatment of the patient designed to observe a temporary depletion of the macrophages making it possible to enhance the transfection rate. Such a technique is described in the literature; see in particular Van Rooijen et al., 1997, TibTech, 15, 178-184.
The invention relates to a cell transfected with a complex as defined above, particularly a prokaryotic cell, a yeast cell or eukaryotic cell, especially an animal cell, in particular a mammalian cell, and more particularly a cancer cell. According to a preferred case of the invention, said cell is a cell of the airways, more particularly a tracheal or pulmonary cell, and advantageously a cell of the respiratory epithelium.
Finally, the invention relates to a device as well as a process allowing the isolation of a molecule of interest containing at least one negative charge, in particular nucleic acids as defined according to the present invention. xe2x80x9cIsolationxe2x80x9d is understood to designate the separation, detection, enrichment and purification of a fraction of anionic molecules, according to a specific or aspecific method of isolation, qualitatively and/or quantitatively.
More particularly, the invention relates to a said device consisting of a disperse solid support, such as for example particles of polymers (of polystyrene, acrylamide, methacrylamide, or of one of their derivatives or any other polymer capable of forming particles of which numerous examples are described in the literature, in particular in the literature relating to diagnostic applications) or consisting of a nondispersed solid support such as, for example, a tube, for example made from polystyrene, a column, for example made from hydroxyapatite, a reversed phase column or an equivalent, onto which at least one compound or one composition according to the invention is bound in its cationic form. The binding to said solid support may be achieved in a direct (adsorption for example) or indirect (via a ligand/anti-ligand type coupling) manner. The production of such devices is within the capability of persons skilled in the art.
Moreover, the invention relates to a process using such a device so as to allow the isolation of anionic molecules, in particular of nucleic acids. Such an isolation may in particular be nonspecific or preferably specific. In this second case, said compound or said cationic composition is, prior to the isolation step, brought into contact with, for example, an oligonucleotide whose specific sequence makes it possible, after a hybridization step, under conditions allowing specific hybridization, to isolate in a specific manner a nucleic acid fragment containing all or part of a sequence complementary to the sequence of said oligonucleotide. The implementation of such a process is widely described in the literature.